Subepithelial fibroblasts form a cellular network just under the epithelium of the gastrointestinal tract. Using primary cultured cells isolated from rat duodenal villi, we previously found that subepithelial fibroblasts reversibly changed cell morphology between flat and stellate-shape depending on intracellular cAMP levels. In this paper, we examined cell-cell communication via released ATP and Ca2+ signaling in the cellular network. Subepithelial fibroblasts were sensitive to mechanical stress such as ;touching' a cell with a fine glass rod and ;stretching' cells cultured on elastic silicone chamber. Mechanical stimulations evoked Ca2+-increase in the cells and ATP-release from the cells. The released ATP activated P2Y receptors on the surrounding cells and propagated Ca2+-waves through the network. Concomitant with Ca2+-waves, a transient contraction of the network was observed. Histochemical, RT-PCR, western blotting and Ca2+ response analyses indicated P2Y1 is a dominant functional subtype. ATP-release and Ca2+ signaling were cell-shape dependent, i.e. they were abolished in stellate-shaped cells treated with dBcAMP, and recovered or further enhanced in re-flattened cells treated with endothelin. The response to ATP also decreased in stellate-shaped cells. These findings indicate cAMP-mediated intracellular signaling causes cell-shape change, which accompanies the changes in mechano- and ATP sensitivities. Using a co-culture system of neuronal cells (NG108-15) with subepithelial fibroblasts, we confirmed that mechanically induced Ca2+-waves propagated to neurons. From these findings we propose that subepithelial fibroblasts work as a mechanosensor in the intestine. Uptake of food, water and nutrients may cause mechanical stress on subepithelial fibroblasts in the villi. The ATP released by mechanical stimulation elicits Ca2+-wave propagation through the network via P2Y1 activation and also activates P2X on terminals of mucosal sensory neurons to regulate peristaltic motility.